The molecular mechanism of action of general anesthetics in the central nervous system is hard to define, but anesthetics are believed to act by preventing synaptic transmission. Anesthetics do act on peripheral synapses and we propose to continue to study their effects at the nicotinic receptor membrane of Torpedo (nAcChoR), which offers the opportunity to relate changes in the receptor's function to changes in its membrane structure. The overall hypothesis is that the mechanisms of action of general anesthetics on the nAcChoR vary with he receptor's conformational state and with the gross structure of the general anesthetics. Pressure will reverse those actions that correlate with lipid solubility, those that have a more specific action will unaffected. The conformational states to be examined are the open channel state, which we hypothesize is inhibited by binding to a hydrophobic site on the protein by some, but not all, general anesthetics, the fast desensitized state, which has not been examine previously, and the slow desensitized state, which we hypothesize is stabilized by perturbation of lipid- protein interactions. Agonist-induced cation efflux from nAcChoR-rich vesicles will be measured on a millisecond time scale to determined which general anesthetics occupy the 1-octanol site we have recently characterized using a flux competition assay. Any agents that do not interact with the 1-octanol site will be examine against each other to determine if further sites are involved. Do different classes of general anesthetics have different sites? The location of this site on the nAcChoR's primary structure will be examine in collaboration with Project 0026. 1. Flux experiments will establish which general anesthetics compete with the photolabel TID 3-Trifluoroethyl-3-(m- iodophenyldiazirine). Time-resolved (millisecond) photolabeling will lead to the identification of amino acid residues associated with channel inhibition. We will establish the underlying kinetics of general anesthetic-induced slow and fast desensitization, and compare these to changes in lipid-protein interactions. Specifically, we will use nAcChoRs reconstituted into bilayer of known composition to test the hypothesis that general anesthetics interact with nonannular cholesterol sites on the nAcChoR. A broad range of general anesthetics will be examined, ranging from alcohols and volatile anesthetics through barbiturates and steroids.
